PROJECT SUMMARY/ABSTRACT Calcium/calmodulin-dependent kinase II (CaMKII) is a Ser/Thr kinase, which plays a critical role in neuronal and cardiac signaling. Unregulated / perturbed activation of CaMKII leads to several pathological conditions, like severe impairment in learning and memory, cardiac arrhythmias and heart failure. Owing to the physiological importance of the activated state of CaMKII, it is obvious that the activation of CaMKII is tightly regulated in cells. My proposed project aims at understanding the molecular basis of this regulation from three perspectives: (1) It is interesting to note that the sequence of this Ca2+ responsive kinase is highly conserved in chordates, except an intrinsically disordered linker that connect the kinase domains to a central dodecameric/tetradecameric hub. Based on the length/composition of this linker, CaMKII is classified into four distinct isoforms and exhibits tissue-specific expression and specific subcellular localization. This intrinsically disordered linker is hypothesized to tune the ease of CaMKII activation in response to varying amplitude/frequency of Ca2+ signals. I will study the role of this intrinsically disordered linker in regulating the activation of CaMKII at a single molecule level. (2) We have recently discovered a unique phenomenon in CaMKII called subunit-exchange, whereby activated CaMKII subunits can exchange into unactivated CaMKII. This can be a mechanism to potentiate activation signal of CaMKII, long after the withdrawal of the initial Ca2+ stimulus. I will study the effect of blocking subunit exchange in cells and eventually I plan to establish a connection between spread of CaMKII activation signal and long-term potentiation/learning/memory. (3) Finally, I will reconstitute the regulatory circuit of CaMKII, including its membrane-associated interaction partners, such as the NMDA receptor, the dopaminergic D3 receptor and the phosphatases, on a supported membrane. Using, this simplistic model, I will study the effect of various signal inputs to this circuit to examine the regulatory constraints on CaMKII at the synapse. I need additional training in microscopy, mass spectrometry and electrophysiological measurements to address these questions. I have assembled a team of mentor and collaborators who will provide me with the technical and intellectual advice for my career development. My short-term (K99/early R00-phase) goal is to apply the newly learned techniques to answer pertinent questions related to the activation and regulation of CaMKII and its role in long-term potentiation, learning and memory. This will position me well to pursue my long- term goal to probe mental health development and maintenance from a molecular perspective. Specifically, I want to investigate the molecular mechanism underlying Down syndrome, focusing on the activation, regulation and downstream signaling effects of a Down syndrome critical region kinase DYRK1a, a promising drug target for Down syndrome neuropathologies.